| ObjectiveGastric cancer(GC)is one of the fatal malignancies worldwide with high occurrences but poor outcomes,is the fifth most common malignant tumor in the world,and is the third leading cause of death in the world after lung cancer and liver cancer.Chemotherapy is the main treatment for advanced gastric cancer.Although great progress has been made in the diagnosis and treatment of gastric cancer,the prognosis of GC patients in advanced stages is still unsatisfied.At present,there is no internationally accepted standard chemotherapy regimen for advanced gastric cancer.Therefore,it is an important research direction of gastric cancer to change the current drug strategy and find more effective combination chemotherapy.MicroRNAs(miRNAs)are a class of non-coding RNA with an endogenous group of21-25nt.They are widely distributed in eukaryotic organisms and conserved in evolutionary sequences.They participate in biological processes,including cell proliferation,differentiation,apoptosis and signal transduction.MicroRNA-195-5p(miR-195-5p)is a kind of microRNAs discovered in recent years.Previous studies have shown that miR-195-5p is abnormally expressed in breast cancer,osteosarcoma and colorectal cancer,and involved in the regulation of apoptosis and formation of tumor cells.However,due to the diversity of the genes encoding the targeted regulatory proteins of microRNAs,the mechanism of how miRNA-195-5p play a role in gastric cancer needs to be further studied.Basic fibroblast growth factor(bFGF)is a kind of polypeptide growth factor with relative molecular weight of(1.6-1.8)*10~4,which can promote the proliferation of cells from mesoderm and neuroectoderm.It is named as basic fibroblast growth factor because it can stimulate the division and proliferation of fibroblasts and the isoelectric point is alkaline(pH 9.6).It has a wide range of biological activities,such as promoting angiogenesis,wound healing,neurotrophic and repair,and embryonic development and differentiation,and is closely related to the occurrence and development of tumor.It is shown that bFGF plays an important role in the occurrence and development of gastric cancer,but there are few studies in mechanism of effect of bFGF in gastric cancer.In this study,we aimed to evaluate the relationship of the expression of microRNA195-5p and bFGF mRNA in gastric cancer,and further identify whether bFGF is a direct target of miR-195-5p in the regulation of tumor progression.MethodsDatebase Target Scan Human or ComiR was retrieved to search for the potential target of miR-195-5p.MicroRNA assays,qPCR and Western blot were conducted for expression analysis of miR-195-5p and basic fibroblast growth factor(bFGF).MiR-195-5p mimics and control mimics were used to transfect gastric cancer cell lines SNU-1 and KATO-3.Two GC cell lines,SNU-1 and KATO-3 overexpressing miR-195-5p and bFGF were subjected to wound healing assay and transwell invasion assay.Luciferase activity was measured with mutated bFGF 3’-UTR sequence at the 3’end of the luciferase gene.The migration and invasion activities of GC cells were evaluated after the transfection of bFGF overexpressing vectors together with miR-195-5p mimics.Mouse GC xenograft model was established.On day 20 the mice were sacrificed and the tumors were dissected,weighed and photographed.Tumor tissues were fixed for further analysis.Results(1)Expression of miR-195-5p and bFGF in human gastric carcinoma was negatively correlated with the level of microRNA sequencing data(r=-0.735,P<0.001);(2)The expression of bFGF in gastric cancer cells decreased in SUN-1 and KATO-3 cell lines with high expression of miR-195-5p;(3)The results of wound healing assay and cell invasion assay of SNU-1 and KATO-3 showed that the migration distance of SNU-1and KATO-3 cell lines overexpressing miR-195-5p at 24 h after scratch was shorter than that of control group.SNU-1 and KATO-3 cell lines overexpressing miR-195-5p significantly reduced the number of invasive cells.These data indicated that miR-195-5p inhibited the migration and invasion of gastric cancer cells.(4)The results of luciferase activity assay showed that the level of miR-195-5p expression in gastric cancer cell line was increased,and the overexpression of miR-195-5p decreased the luciferase activity of bFGF WT 3’-UTR when the two luciferase vectors were stably expressed in gastric cancer cell line.However,the luciferase activity of bFGF-MUT3’-UTR was not changed,which confirmed that miR-195-5p directly recognized and targeted bFGF 3’-UTR and inhibited the expression of bFGF.(5)The high expression of miR-195-5p in gastric cancer transplantation model was established.The data showed that tumor weight and volume were significantly inhibited in the miR-195-5p group compared with the control group.(6)In animal experiments,the expression of mRNA and protein of bFGF in tumor tissues was inhibited in both SNU-1 and KATO-3 models with high expression of miR-195-5p,which was consistent with our early experimental data in vitro.This suggests that miR-195-5p can down-regulate the protein expression of basic fibroblast growth factor in vitro and in vivo.The expression of mRNA and protein in SNU-1 and KATO-3 cells after overexpression of miR-195-5p and bFGF in both gastric cancer cells returned to the control level.(7)After overexpression of miR-195-5p in SNU-1 and KATO-3 cells,the ability of cell migration and invasion decreased,and the inhibition could be reversed when bFGF was reintroduced into these two kinds of cells.Similarly,when these cells were injected to the right of SCID mice to establish xenotransplantation models,the expression of bFGF significantly reversed the inhibitory effect of miR-195-5p on tumor size of gastric cancer.ConclusionsMiR-195-5p inhibits tumorigenesis of GC through suppressing bFGF.This may provide a new insight for studying the pathogenesis of gastric cancer and a new potential therapeutic target for advanced gastric cancer(especially in targeted therapy)in future. |
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